Redundant essentiality of AsmA-like proteins in Pseudomonas aeruginosa

ABSTRACT The outer membrane (OM) is an essential structure of Gram-negative bacteria that provides mechanical strength and protection from large and/or hydrophobic toxic molecules, including many antibiotics. The OM is composed of glycerophospholipids (GPLs) and lipopolysaccharide (LPS) in the inner and outer leaflets, respectively, and hosts integral β-barrel proteins and lipoproteins. While the systems responsible for translocation and insertion of LPS and OM proteins have been elucidated, the mechanism(s) mediating transport of GPLs from the inner membrane to the OM has remained elusive for decades. Very recently, studies performed in Escherichia coli proposed a role in this process for AsmA-like proteins that are predicted to share structural features with eukaryotic lipid transporters. In this study, we provide the first systematic investigation of AsmA-like proteins in a bacterium other than E. coli, the opportunistic human pathogen Pseudomonas aeruginosa. Bioinformatic analyses revealed that P. aeruginosa possesses seven AsmA-like proteins. Deletion of asmA-like genes in many different combinations, coupled with conditional mutagenesis, revealed that four AsmA-like proteins are redundantly essential for growth and OM integrity in P. aeruginosa, including a novel AsmA-like protein (PA4735) that is not present in E. coli. Cells depleted of AsmA-like proteins showed severe defects in the OM permeability barrier that were partially rescued by lowering the synthesis or transport of LPS. Since fine balancing of GPL and LPS levels is crucial for OM integrity, this evidence supports the role of AsmA-like proteins in GPL transport toward the OM. IMPORTANCE Given the importance of the outer membrane (OM) for viability and antibiotic resistance in Gram-negative bacteria, in the last decades, several studies have focused on the characterization of the systems involved in OM biogenesis, which have also been explored as targets for antibacterial drug development. However, the mechanism mediating translocation of glycerophospholipids (GPLs) to the OM remained unknown until recent studies provided evidence that AsmA-like proteins could be responsible for this process. Here, we demonstrate for the first time that AsmA-like proteins are essential and redundant for growth and OM integrity in a Gram-negative bacterium other than the model organism Escherichia coli and demonstrate that the human pathogen Pseudomonas aeruginosa has an additional essential AsmA-like protein that is not present in E. coli, thus expanding the range of AsmA-like proteins that play key functions in Gram-negative bacteria.


SUPPLEMENTARY MATERIAL
Table S1.Bacterial strains used in this study.
Table S2.Plasmids used in this study.
Table S4.Primers used to generate the plasmids described in Table S2.
Figure S1.Plating efficiency of the P. aeruginosa mutants with single, double or triple deletions in asmA-like genes that showed defects with respect to the wild type strain.catgccatGGCGAATCGCTTGTGCAAAG NcoI a Preparative PCRs for cloning were performed using the genomic DNA of P. aeruginosa PAO1 as the template.b The restriction site used for cloning is underlined in the primer sequence.

Figure S1.
Plating efficiency of the P. aeruginosa wild type PAO1 and all isogenic mutants with single, double or triple deletions in asmA-like genes that showed defects with respect to the wild type strain on MH agar plates supplemented or not with SDS and EDTA (0.25% and 0.25 mM, respectively), vancomycin (Van) or colistin (Col) at concentrations corresponding to 0.05, 0.25 or 0.5×MIC for the wild type strain (as indicated).
Images were taken after 24 h of incubation at 37°C and are representative of at least three independent experiments.All the other mutants generated in this work (Table S1) and not included in this figure did not show plating efficiency defects under the conditions tested (data not shown).S3).The PA4735 protein is in blue, while the putative PA4735 structural homologs are in yellow.then diluted 1:30 in MH broth (without rhamnose).After 2 h, the cultures were further diluted 1:50 in fresh medium, and growth (OD600) was monitored over time.Values are the mean (± standard deviation) of three independent experiments.Bacterial cells were collected for microscopy and cell envelope stability assays as soon as a growth defect was observed in the conditional mutant with respect to the wild type strain (6 h).

Figure S2 .
Figure S2.Structural alignment of PA4735 with selected putative structural homologs.

Figure S4 .
Figure S4.Culturing strategy used to obtain P. aeruginosa cells depleted in AsmA-like proteins of interest.

Figure S5 .
Figure S5.Confocal microscopy images of selected P. aeruginosa mutants in asmA-like genes expressing a FtsZ-GFP fusion protein.
the DNA regions upstream and downstream of the tamA coding sequence, used for the in-frame deletion of tamA by homologous recombination This study pDM4ΔyhdP pDM4 derivative carrying the DNA regions upstream and downstream of the yhdP coding sequence, used for the in-frame deletion of yhdP by homologous recombination This study pDM4ΔasmA pDM4 derivative carrying the DNA regions upstream and downstream of the asmA coding sequence, used for the in-frame deletion of asmA by homologous recombination This study pDM4ΔydbH pDM4 derivative carrying the DNA regions upstream and downstream of the ydbH coding sequence, used for the in-frame deletion of ydbH by homologous recombination This study pDM4ΔyhjG pDM4 derivative carrying the DNA regions upstream and downstream of the yhjG coding sequence, used for the in-frame deletion of yhjG by homologous recombination This study pDM4ΔPA4735 pDM4 derivative carrying the DNA regions upstream and downstream of the PA4735 coding sequence, used for the inframe deletion of PA4735 by homologous recombination This study pDM4ΔPA2708 pDM4 derivative carrying the DNA regions upstream and downstream of the PA2708 coding sequence, used for the inframe deletion of PA2708 by homologous recombination This study pDM4ΔlptB pDM4 derivative carrying the DNA regions upstream and downstream of the lptB coding sequence, used for the in-frame deletion of lptB by homologous recombination This study pJM253 mini-CTX1 derivative carrying rhaRS-PrhaBAD, Tc R 69 pJM253tamB pJM253 derivative carrying the coding sequence of tamB downstream of PrhaBAD This work pJM253lptB pJM253 derivative carrying the coding sequence of lptB downstream of PrhaBAD This work pME6032 IPTG inducible expression vector, lacI q -Ptac, Tc R 71 pMEftsZ-GFP pME6032 derivative carrying the coding sequence of ftsZ fused to the coding sequence of the GFP gene downstream of the IPTGinducible Ptac promoter 35 pMEyhdP pME6032 derivative carrying the coding sequence of yhdP downstream of the IPTG-inducible Ptac promoter This work pMEydbH pME6032 derivative carrying the coding sequence of ydbH downstream of the IPTG-inducible Ptac promoter This work pMEPA4735 pME6032 derivative carrying the coding sequence of PA4735 downstream of the IPTG-inducible Ptac promoter This work pFLP2 Broad-host-range plasmid expressing the Flp recombinase, sacB; Ap R /Cb R

Figure S3 .
Figure S3.(A) Confocal microscopy images of P. aeruginosa PAO1 and the single deletion mutants in tamB, yhdP, ydbH or PA4735 cultured in MH until the mid-exponential phase and stained with the membranelabelling dye FM4-64.Images are representative of two independent experiments and several fields of view showing the same results.(B) Violin plots showing cell area (μm 2 ) and aspect ratio (length/width) for 100 cells of the same strains shown in panel A, calculated with the ImageJ software.

Figure S4 .
Figure S4.Dual-refresh culturing strategy used to obtain TamB-depleted cells of the conditional mutant ΔyhdP ΔydbH ΔtamB rhaSR-PrhaBAD::tamB ΔPA4735.P. aeruginosa PAO1 and the conditional mutant were cultured overnight in flasks at 200 rpm and 37°C in MH broth supplemented with 0.01% rhamnose (not shown) and

Figure S5 .
Figure S5.Confocal microscopy images of P. aeruginosa PAO1 and the mutants ΔtamB ΔyhdP, ΔtamB ΔyhdP ΔydbH, ΔtamB ΔyhdP ΔPA4735, and ΔyhdP ΔydbH ΔtamB rhaSR-PrhaBAD::tamB ΔPA4735 carrying the plasmid pME ftsZ-GFP, cultured in the presence of 0.003 mM IPTG to induce the expression of the fusion protein FtsZ-GFP at non-toxic levels (35) and stained with the membrane-labelling dye FM4-64.Strains were cultured in MH until the mid-exponential phase, except for the conditional mutant ΔyhdP ΔydbH ΔtamB rhaSR-PrhaBAD::tamB ΔPA4735 that was progressively depleted of TamB using the culturing strategy shown in Figure S4.Images are representative of two independent experiments and several fields of view showing the same results.

Table S1 .
Bacterial strains used in this study.

Table S2 .
Plasmids used in this study.

Table S3 .
Putative structural homologs of PA4735 identified by FoldSeek in the AlphaFold database.a a Only the 10 putative structural homologs with the highest score are described in the table.The other putative structural homologs can be retrieved at https://search.foldseek.com/result/hPOmiVVMLzrWTF9BD4SI1jJlx0OMQQvtG2jcjw/0.

Table S4 .
Primers used to generate the plasmids described in TableS2.a